APR Sept/Oct 2023 - 54

» MICROBIOLOGY
»
transfer rate (where heat and volume are inversely
proportional), control of the gas volumetric flow
rate (which requires control of pressure), and the
maintenance of a constant gas superficial velocity.
* The production of high protein yield.
* Additional use of cell retention devices (such as tangential flow
filtration or spin filters) when the perfusion culture technique is
used.
*
Suitability for use in transfection (to study and modulate gene
expression).
* Cell survival in serum-free media.
* Controls to lower the risk of viral infection.
To enhance production, high-throughput devices can be used for
bioprocess optimization. As with other areas of biopharmaceutical
manufacturing, advances have been made with sterile, disposable
single-use systems, continuous upstream processing, continuous
chromatography, and integrated continuous bioprocessing.
Efficiency can also be delivered at the outset by applying Quality by
Design and through manufacturing by applying process analytical
technologies to achieve the desired quality of the product and the
target yield. Continuous bioprocessing is also being applied for
both upstream and downstream manufacturing, which allows for a
smaller production footprint.
Process Problems
Proteins are sensitive to their surrounding chemical environment.
Changes in pH, ionic strength, and oxidative status can each impact
protein stability. During purification, care needs to be taken with the
stabilization of the target protein and to facilitate tag exposure. This
leads to the control parameters for the bioreactor being important.
This extends to operating parameters like temperature, pH, agitation,
aeration, dissolved oxygen, carbon dioxide, and hydrodynamic shear
forces. Aspects that are more likely to go wrong and adversely affect
the process are temperature shifts and gas exchange.
Other things can go wrong during the manufacturing process. These
include weak growth of the host; inclusion body formation; protein
inactivity; contamination of cell lines (particularly from prions, and
oncogenic DNA); and failure to obtain any protein at all.20
Types of Contamination
Microbial contamination includes viral and mycoplasma infection of
cell lines. Viruses are of concern as common contaminants and because
they are more difficult to detect than other microbial contaminants.
Viral contamination is controlled by:
* The selection of suitable raw materials.
* Testing cell banks and in-process materials.
* Putting in place control measures to remove or to inactivate
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| September/October 2023
potential undetected viral contaminants during the purification
stages.
Raw materials should have a low risk of containing endogenous or
adventitious viruses and intermediate manufacturing steps should
be assessed to ensure the in-process material is free from detectable
viruses. Different viruses will pose different risks to specific cell lines.
Techniques like PCR are optimal for virus screening, provided the
required primers and probes are available.
Mycoplasma contamination is a widespread and reoccurring
problem for many cell culture systems in life science research and
the pharmaceutical industry. Mycoplasmas are bacteria of a size
0.15-0.3 μm in diameter and they can grow to high titers in culture
media without exhibiting typical bacterial contamination signs such
as turbidity. The deleterious effects on cultured cells include fusing
with and/or invading the host cell, inducing severe cytopathic effects,
causing abnormal cell growth, altering the host's gene expression
profile (i.e., oncogenes, tumor suppressor genes, cytokines, and
signaling regulators), outcompeting the host for culture nutrients,
including sugars, heavy metals, or amino and fatty acids; causing
slowed proliferation, and chromosomal aberrations. As a result,
mycoplasma testing and the maintenance of contamination-free cell
cultures is essential for manufacturing.
Ironically, one of the contaminants can be endotoxin. Bacterial
endotoxin can readily contaminate water, aqueous solutions, and
buffers, and therefore suitable controls are required over the quality of
water and reagents used during manufacturing. The main challenge
posed by endotoxin is with its binding affinity to different protein
surfaces.21
To treat the prepared protein, commonly used techniques
for removing endotoxin contaminants are ultrafiltration and ion
exchange chromatography.
One source of non-microbial contamination can relate to bioreactors,
where metal ions such as iron, chromium, and nickel can exist as
leachables under different formulation buffer and pH conditions.
With non-microbial contamination, the primary challenge is from the
lack of any standardized assay with sufficient sensitivity to cover all
possible contaminants of the end-product. Data signaling a concern
is often provided retrospectively, such as from a customer complaint
indicating a potential aberrant release or from a stability result.
Process Optimization and Scale-Up
Manufacturers will also invest heavily in process optimization, devising
strategies to increase product yield. When scaling up between
development and larger scale manufacturing, control of parameters
is especially important. Successful scale-up is measured by sufficient
cell growth, maintaining cell viability, and the achieved protein
production (the titer). Given the scale of the endotoxin testing industry
and the expected expanding demand for recombinant endotoxin
test reagents, consistently manufacturing high-purity recombinant
products to scale represents an on-going priority.
APR Sept/Oct 2023

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